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Endocrinology Vol. 147, No. 4 1950-1958
Copyright © 2006 by The Endocrine Society

Redundant and Synergistic Effect of Cdx-2 and Brn-4 on Regulating Proglucagon Gene Expression

Peixiang Wang, Tao Liu, Zhihong Li, Xuezhong Ma and Tianru Jin

Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network; and Departments of Medicine, Laboratory Medicine, and Pathobiology, Banting and Best Diabetes Center, University of Toronto, Toronto, Ontario, Canada M5G 2M1

Address all correspondence and requests for reprints to: Dr. Tianru Jin, Room 410, 67 College Street, Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada M5G 2M1. E-mail: tianru.jin{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cdx-2 and Brn-4 are recognized as transcriptional activators for the proglucagon gene. These two homeodomain (HD) proteins are able to interact with the correspondent motifs on the G1 enhancer element of proglucagon promoter, separated by only 8 bp. We have examined Brn-4 expression in proglucagon-producing cells, isolated hamster Brn-4 cDNA, and localized its activation domain. Ectopic expression of either Cdx-2 or Brn-4 in the pancreatic B cell line In111 provoked it to express proglucagon mRNA, whereas ectopically expressing both of them further stimulated proglucagon mRNA expression in this cell line. Furthermore, Brn-4 was found to synergize with Cdx-2 in activating proglucagon promoter, and the Brn-4 activation domain was not required for this synergistic activation. When the binding site for either Cdx-2 or Brn-4 was mutated, the synergistic activation by these two HD proteins was significantly attenuated, but not abolished. We propose that both cooperative DNA binding and mutual recruitment between Cdx-2 and Brn-4 are involved in this synergistic activation and have detected physical interaction between Cdx-2 and Brn-4 by glutathione-S-transferase-fusion protein pull-down assay. Our observations suggest that Cdx-2 and Brn-4, two HD proteins that belong to two different families, exert a synergistic and redundant effect on proglucagon gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROGLUCAGON gene is expressed in pancreatic islet A cells, intestinal endocrine L cells, and selected neuronal cells in the brain. Proglucagon mRNA encodes three major peptide hormones, glucagon, glucagon-like peptide 1 (GLP-1), and GLP-2 (1, 2). These peptide hormones [proglucagon-derived peptides (PGDPs)] may have opposite or overlapping physiological functions. Glucagon produced in pancreatic islet A cells is a major counterregulatory hormone to insulin in blood glucose homeostasis. GLP-1 and GLP-2, however, are synthesized in intestinal endocrine L cells and in selected endocrine neurons in the brain. GLP-1 stimulates insulin secretion, inhibits glucagon release, and enhances peripheral insulin sensitivity (2). In addition, intracerebroventricular administration of GLP-1 was found to powerfully inhibit eating and drinking and to alter body weight in fasted rats (3, 4). Furthermore, a recent study has shown that peripheral GLP-1 also plays a role in regulating macronutrient selection and food intake (4). GLP-2 was initially identified as a growth factor for small intestinal epithelia (5). Recent studies suggested that GLP-2 may possess overlapping function with GLP-1 in controlling gastric emptying and satiety (6).

Extensive studies have been conducted to examine the mechanisms that control proglucagon expression and the genesis of proglucagon-producing cells (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). These studies have identified a number of cis elements on the proglucagon gene promoters, and more than a dozen transcription factors and signaling molecules that are implicated in proglucagon expression. Among the transcription factors identified, several of them are homeodomain (HD) proteins, including the caudal HD protein Cdx-2 and the POU (Pit-1, Oct-1 and Oct-2, Unc-86) HD protein Brn-4 (12, 15). These two HD proteins were shown to bind to the motifs on the evolutionarily conserved proglucagon gene promoter G1 enhancer element, separated by only 8 bp (12, 15). In this study we examined the expression of Brn-4 in the proglucagon-producing cell line, isolated hamster Brn-4 cDNA, and investigated the redundant and synergistic roles of Cdx-2 and Brn-4 in regulating proglucagon promoter and mRNA expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture medium, fetal bovine serum, and oligonucleotides were purchased from Invitrogen Life Technologies, Inc. (Burlington, Canada). Radioisotopes were obtained from Amersham Biosciences (Baie d’Urfe, Canada). Restriction enzymes and DNA modification enzymes were molecular biology grade and were purchased from several sources.

Plasmids, RNA extraction, RT-PCR, and DNA sequencing
Construction of the proglucagon/luciferase (GLU-LUC) reporter gene plasmids has been previously described (14). Two plasmids of the Gal4-LUC reporter system [pFA/Gal4-LUC and pFR-cytomegalovirus (CMV)] were purchased from Stratagene Corp. (La Jolla, CA). Total RNAs from cultivated cell lines and the mouse tissues were extracted using TRIzol reagent purchased from Invitrogen Life Technologies, Inc. Hamster Brn-4 cDNA was isolated from a pancreatic islet A cell line InR1-G9 by RT-PCR, using the Vent polymerase (New England BioLabs, Beverly, MA). It was then inserted into the pCDNA3.1 expression vector. Three deletion constructs of Brn-4 (D1, D2, and D3; Fig. 1BGo) were then generated by PCR, with the pCDNA3-Brn-4 as the template, using the Vent polymerase. The PCR products were inserted into the same pCDNA3.1 expression vector. Nucleotide sequences of the hamster Brn-4 were determined by DNA sequencing for both strains. Four additional deletion constructs of Brn-4 (NT1, NT2, HD, and CT) were generated for localization of the Brn-4 activation domain with the Gal4-LUC reporter gene system and for identification of the Cdx-2 interaction domain with the glutathione-S-transferase (GST) fusion protein pull-down assay (Fig. 1BGo). The oligonucleotide primers used in RT-PCR and PCR are shown in Table 1Go.


Figure 1
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  		FIG. 1. A, DNA sequence of the rat proglucagon gene promoter G1 enhancer element. The Brn-4-binding site and two Cdx-2-binding sites are underlined. This region is conserved between proglucagon genes of humans and rodent species. B, Schematic illustration of hamster Brn-4 and its deletion mutants generated for this study. POU, POU DNA-binding domain; NT, N terminus; CT, C terminus.

 

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  		TABLE 1. Oligonucleotides/primers used in RT-PCR and PCR

 
The wild-type (WT) GLU(G1)-thymidine kinase (TK)-LUC and Cdx-2M or Brn-4M GLU(G1)-TK-LUC fusion gene constructs used (see Fig. 8Go) were constructed as follows. The WT (or Cdx-2M) or Brn-4M G1 element was generated by annealing a pair of correspondent oligonucleotides (Fig. 1AGo). It was then inserted into the TK-LUC plasmid (15) via the HindIII and BamHI restriction sites. The final products were verified by automatic DNA sequencing.


Figure 8
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  		FIG. 8. Mutating the Cdx-2- or Brn-4-binding site attenuated, but did not abolish, the synergistic activation. BHK fibroblasts were transfected with 2 µg of the indicated GLU-TK-LUC (WT, Cdx-2M, or Brn-4M) and the indicated amount (micrograms) of Brn-4 or Cdx-2 cDNA or with pCDNA3.1 (as vector). Cells were harvested for LUC reporter gene analysis 18 h after transfection. Relative LUC reporter gene activity was calculated as the fold increase in activity compared with that in cells that received the empty pCDNA3.1 plasmid transfection, which was defined as 1-fold (mean ± SE; n = 3).

 
Cell culture, transfection, and LUC reporter gene analysis
Hamster pancreatic cell lines InR1-G9 and In111, mouse pancreatic cell line RIN-1056A, mouse intestinal cell lines GLUTag and STC-1, the baby hamster kidney (BHK) fibroblasts, and the COS-1 cell line were grown and maintained in DMEM supplemented with the appropriate serum as described previously (15). To generate Cdx-2-expressing In111 cell clones, hamster Cdx-2 cDNA (27) was inserted into the pCDNA3.1-expressing vector and transfected into the In111 cell line using Lipofectamine (Invitrogen Life Technologies, Inc.). The same approach was used to generate Brn-4-expressing In111 cell clones. Positive clones were selected using G418 and were verified by Western blotting using either anti-Cdx-2 or anti-Brn-4 antibodies (28).

For LUC reporter gene analysis, cell line transfection was conducted by calcium precipitation (14). Sixteen to 18 h after transfection, cells were harvested for LUC reporter gene analysis as previously described (14).

GST-fusion protein pull-down assay
The WT GST-Brn-4 fusion protein construct and the deletion mutant constructs (D1, D2, D3, and HD; Fig. 1BGo) were generated by inserting the correspondent coding sequence of Brn-4 into the pGEX4T-2 vector (Amersham Biosciences). The fusion gene plasmids were then transformed into the BL-21 strain of Escherichia coli. Expression of the GST fusion protein was induced by 0.3 µM isopropyl-ß-D-thiogalactoside and purified using the glutathione resin (Amersham Biosciences). These proteins were then used in the pull-down assay against whole cell lysate from Cdx-2-transfected COS-1 cells. The presence of Cdx-2 was detected by Western blotting. Part of the GST or GST-Brn-4 fusion proteins coupled to the glutathione resin was eluted with SDS sample buffer and examined by 10% SDS-PAGE.

Northern and Western blotting analyses
Methods for Northern and Western blotting analyses have been reported previously (15, 28). The rabbit polyclonal antibody against a C-terminal portion of hamster Cdx-2 (amino acids 260–313) was generated in our previous studies (29); the anti-Brn-4 antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The antiactin antibody was purchased from Sigma-Aldrich Canada Ltd. (Oakville, Canada).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of hamster Brn-4 cDNA
Brn-4 has been shown to bind to an A-T-rich motif within the G1 enhancer element of the proglucagon gene promoter, 8 bp upstream of the major Cdx-2-binding site (Fig. 1AGo) (20). Based on the known cDNA sequence information for rat and mouse Brn-4, a pair of primers (Brn-4F and Brn-4R; see Materials and Methods and Table 1Go) were designed and used to isolate hamster Brn-4 cDNA from the pancreatic A cell line InR1-G9 by RT-PCR. The expression of Brn-4 in this cell line has been previously demonstrated by Hussain et al. (12) by Western blot analysis. A single fragment of approximately 1.1 kb was obtained. This fragment was then inserted into a ZeroBlunt vector (Invitrogen Life Technologies, Inc.), followed by DNA sequencing using a manual method for both strands. The coding region of the hamster Brn-4 cDNA shares 96% nucleotide sequence identity with that of the mouse Brn-4 cDNA, whereas the amino acid sequence identity is as high as 99% (GenBank accession no. AF469664). Human Brn-4 cDNA shares 91% nucleotide sequence identity and 98% amino acid sequence identity with that of the hamster Brn-4.

For the Brn-4 protein, the two DNA-binding domains, i.e. the POU-specific DNA-binding domain and the POU HD domain, are separated by a 15-amino acid residue linker (Fig. 1BGo). The N and the C termini are 184 and 27 amino acid residues in length, respectively (Fig. 1BGo).

Examination of Brn-4 expression
We found previously that the pancreatic B cell line In111 does not express Cdx-2 and proglucagon mRNA (15). To examine Brn-4 expression in this and other cell lines and in mouse tissues, RT-PCR was used. Figure 2AGo shows that Brn-4 expression could be detected by RT-PCR in mouse pancreas, brain, InR1-G9, GLUTag, and STC-1 cell lines, but not in mouse liver and In111 cell lines. The rat RIN-1056A cell line, which express both proglucagon and insulin, does not express a detectable amount of Brn-4 mRNA. The expression of Brn-4 protein has been shown previously by Hussain et al. (12) in the proglucagon-producing intestinal GLUTag and pancreatic InR1-G9 cell lines. By Western blotting we demonstrate here that Brn-4 protein is also expressed in another intestinal proglucagon-producing cell line, STC-1, but not in the insulin-producing In111 cell line or the RIN-1056A cell line (Fig. 2BGo). Figure 2BGo also shows the expression of Cdx-2 protein in STC-1, InR1-G9, and RIN-1056A cell lines, but not in the In111 cell line. Therefore, Cdx-2 is expressed in all proglucagon-expressing cell lines, whereas lack of Brn-4 expression is observed in both proglucagon-expressing (RIN-1056A) and nonexpressing (In111) cell lines.


Figure 2
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  		FIG. 2. Detection of Brn-4 expression. A, Detection of Brn-4 mRNA expression by RT-PCR in pancreatic and intestinal endocrine cell lines and mouse tissues. –, No cDNA was added. Detection of actin expression serves as the control for the quality of cDNA generated for each sample. B, Detection of Brn-4 and Cdx-2 protein expression in four pancreatic and intestinal endocrine cell lines. Cell lysates were prepared and analyzed for Cdx-2 expression by Western blotting. The same membrane was stripped and rehybridized against anti-Brn-4 and anti-ß-actin antibodies.

 
Ectopic Brn-4 expression provoked the In111 cell line to express proglucagon mRNA
Considering the observations made by Hussain et al. (19, 20, 21), we wondered whether we would be able to use the WT In111 cell line to examine the effect of ectopic Brn-4 expression on proglucagon gene expression. For this purpose we have generated stably transfected In111 cell clones with Brn-4 cDNA, inserted into the pCDNA3.1 vector (designated the In111-Brn-4 clone). Among 12 individual clones we have isolated, six were found to express a detectable amount of Brn-4 protein. Figure 3AGo shows our representative Western blotting results for the expression of Brn-4 protein in these six clones and in the culture pool, which was collected after the antibiotic selection procedure, before the generation of single-cell-derived clones. By conducting Northern blotting analysis, we found that, except for clone 5, all the other five In111-Brn-4 clones express a detectable amount of proglucagon mRNA (Fig. 3BGo). However, none of the other six clones, which do not express Brn-4 protein, was found to express proglucagon mRNA (data not shown). Therefore, ectopic expression of Brn-4 rendered the pancreatic B cell line In111 to express proglucagon mRNA.


Figure 3
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  		FIG. 3. Effect of ectopic Brn-4 expression on endogenous proglucagon mRNA expression in the In111 cell line. A, Brn-4 protein expression in the In111-Brn-4 clones was detected by Western blotting. The same membrane was stripped and rehybridized against the anti-ß-actin antibody. The WT In111 cell line was used as the negative control, and STC-1 cell line was used as the positive control. The pool represented In111 cell culture after the Brn-4 cDNA transfection and antibiotic selection, before the single-clone selection procedure. B, Approximately 10 µg total RNA from the indicated cell lines/clones was applied to Northern blotting for examination of endogenous proglucagon mRNA (glu) expression. The same membrane was stripped, followed by hybridization with the tubulin probe (Tub).

 
Ectopic Cdx-2 expression provoked the In111 cell line to express proglucagon mRNA
As presented in Fig. 2BGo and in our previous studies (23), the In111 cell line does not express Cdx-2. Intrigued by the fact that Brn-4 can induce proglucagon mRNA expression in this cell line to some extent, we wondered whether Cdx-2 would also be able to trigger its proglucagon gene expression.

We then stably transfected WT In111 cells with the hamster Cdx-2 cDNA. Among 12 individual clones we screened, six were shown to express a detectable amount of Cdx-2 protein by Western blotting (designated In111-Cdx-2 clone), although the expression level was not as high as in the InR1-G9 cell line (Fig. 4AGo). As shown in Fig. 4BGo, although the WT In111 cell line does not express proglucagon mRNA (lane 2), four of the six In111-Cdx-2 clones expressed detectable amounts of proglucagon mRNA by Northern blotting (clones 2, 3, 5, and 6; Fig. 4BGo). None of the other six clones, which do not express Cdx-2, was found to express proglucagon mRNA (data not shown). Therefore, ectopic expression of Cdx-2 in the In111 cell line also provoked it to express proglucagon mRNA.


Figure 4
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  		FIG. 4. Effect of ectopic Cdx-2 expression on endogenous proglucagon mRNA expression in the In111 cell line. A, Cell lysates from WT InR1-G9, WT In111, and six individual In111-Cdx-2 clones were prepared and analyzed for Cdx-2 expression by Western blotting. The same membrane was stripped and rehybridized against the anti-ß-actin antibody. B, Approximately 10 µg total RNA from the indicated cell lines/clones were applied to Northern blotting for examination of endogenous proglucagon mRNA (glu) expression. The same membrane was stripped, followed by hybridization with the tubulin probe (Tub).

 
Ectopic Cdx-2 and Brn-4 expression further stimulated proglucagon mRNA expression
We have made attempts to stably transfect both Cdx-2 and Brn-4 into the In111 cell line without success. We therefore transiently transfected Brn-4 cDNA into In111-Cdx-2 clone 2 and examined the effect of ectopic expression of these two HD proteins on proglucagon mRNA expression. A representative result is shown in Fig. 5Go. Neither the WT In111 cell line nor In111-Cdx-2 clone 2 expressed Brn-4 protein (Fig. 5AGo, lanes 1 and 2), whereas after Brn-4 cDNA transient transfection, Brn-4 protein expression could be detected (Fig. 5AGo, lanes 3 and 4). It appears that Brn-4 expression did not affect Cdx-2 expression in In111-Cdx-2 clone 2 (comparing lanes 2, 3, and 4). Figure 5BGo shows that transient Brn-4 cDNA transfection further stimulated proglucagon mRNA expression in In111-Cdx-2 clone 2. Taken together, our observations suggest that Cdx-2 and Brn-4 may exert both redundant and synergistic effects on proglucagon mRNA expression.


Figure 5
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  		FIG. 5. Effect of ectopic Cdx-2 and Brn-4 expression on endogenous proglucagon mRNA expression in the In111 cell line. A, Cell lysates from WT In111, In111-Cdx-2 (C2), and In111-Cdx-2 (C2) cells transfected with 5 or 10 µg Brn-4 were prepared and analyzed for Brn-4 and Cdx-2 expression by Western blotting. B, Approximately 10 µg total RNA from the indicated cell lines/clones was applied to Northern blotting for examination of endogenous proglucagon mRNA (glu) expression. The same membrane was stripped, followed by hybridization with the tubulin probe (Tub).

 
Localization of the activation domain of Brn-4
To initiate the exploration of molecular mechanisms underlying the potential synergistic activation on proglucagon gene expression by Cdx-2 and Brn-4, we localized the activation domain of Brn-4. The BHK fibroblast naive cell system, which does not express proglucagon, Brn-4, and Cdx-2 (15), was used in this study. When the WT or the three deletion Brn-4 expression plasmids (Fig. 1BGo) were cotransfected with the –82GLU-LUC fusion gene into BHK cells, no substantial change in LUC reporter gene expression was observed (Fig. 6AGo, left panel). This is not unexpected, because this reporter gene construct does not contain the Brn-4-binding site (Fig. 1AGo). When the same set of experiments was conducted using the –155GLU-LUC fusion gene, WT Brn-4 activated the reporter gene expression more than 3.5-fold. However, none of the Brn-4 deletion mutants had any stimulatory effect (Fig. 6AGo, right panel). This is consistent with a previous report that the binding site for Brn-4 is ATTATTTA (Fig. 1AGo), upstream of –82 bp (12). This observation would also suggest that the activation domain of Brn-4 is located within the first 89 amino acid residues (Fig. 1BGo).


Figure 6
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  		FIG. 6. Localization of the Brn-4 activation domain. A, BHK fibroblasts were transfected with 3 µg of the indicated GLU-LUC [as reporter (Rep.)] and 3 µg of the indicated cDNA plasmid (WT Brn-4 or its deletion mutants, WT, D1, D2, and D3) or with 3 µg empty vector (V; pCDNA3.1). B, BHK fibroblasts were transfected with 3 µg of the indicated Gal4-LUC [as reporter (Rep.)] and 3 µg of the indicated cDNA plasmid (Gal4-Brn-4 deletion mutants) or with 3 µg empty vector (pFR-CMV). C, InR1-G9 cells were transfected with 3 µg of the indicated GLU-LUC [as reporter (Rep.)] and 3 µg of the indicated cDNA plasmid (Cdx-2, Brn-4, or Brn-4 deletion mutants) or with 3 µg empty vector (V; pCDNA3.1). Cells were harvested for LUC reporter gene analysis 18 h after transfection. Relative LUC reporter gene activity was calculated as the fold increase in activity compared with that in cells that received empty vector (V) transfection, which was defined as 1-fold (mean ± SE; n = 3).

 
To confirm that the transcriptional activation domain of Brn-4 is indeed located within its N terminus, we inserted the DNA sequences that encode the amino acid residues of 1–89 (NT1), 1–185 (NT2), and 321–361 (CT) of Brn-4 into the pFR-CMV plasmid, which is a Gal4 fusion expression vector (Stratagene Corp.). These three Gal4 fusion constructs were then cotransfected into BHK cells along with the reporter gene pFA/Gal4-LUC (Stratagene Corp.). As shown in Fig. 6BGo, although pRF-CMV-Brn-4-(1–89) and pRF-CMV-Brn-4-(1–185) activated Gal4-LUC reporter gene expression approximately 5- and 7-fold, respectively; pRF-CMV-Brn-4-(321–361) generated no stimulatory effect. Taken together, our results suggest that the main activation domain of Brn-4 is located within amino acid residues 1–89.

We also conducted a LUC reporter gene assay in the Cdx-2- and Brn-4-expressing InR1-G9 cell line. As shown in the left panel of Fig. 6CGo, WT Brn-4 cDNA transfection activated the expression of –155GLU-LUC about 2-fold, much less potent than Cdx-2 transfection (~8-fold; right panel). The D2 and D3 deletion mutants of Brn-4 generated virtually no stimulatory effect. Interestingly, the D1 mutant reproducibly repressed reporter gene activity by approximately 65%. This repressive function of D1 was not observed when BHK (Fig. 6GoA) and COS-1 (data not shown) cells were examined, suggesting that D1 could serve as a dominant-negative molecule in proglucagon-producing cells. One possible explanation is that a high dosage of Brn-4 interferes with the function of other transcriptional activators. To examine this possibility, we conducted the experiments and observed that high dosages of Brn-4 repressed the proglucagon promoter in the InR1-G9 cell line, in contrast to the fact that Brn-4 activated the expression of –155GLU-LUC in BHK cells dose dependently (data not shown).

Brn-4 synergizes with Cdx-2, and the Brn-4 activation domain is not required for its synergistic capability
After identification of the Brn-4 activation domain, we investigated whether Brn-4 and Cdx-2 synergistically activate proglucagon promoter using the BHK naive cell system (15, 29). As shown in Fig. 7Go, although no synergistic activation on –82GLU-LUC expression was observed by Cdx-2 and Brn-4 cotransfection (A), substantially synergistic activation was observed when –155GLU-LUC was examined (B), indicating that the Brn-4-binding site is required for this synergistic activation. More interestingly, the D1 deletion mutant of Brn-4 also synergized with Cdx-2 in activating –155GLU-LUC expression, although less potently, indicating that the Brn-4 activation domain is not absolutely necessary for the synergistic activation.


Figure 7
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  		FIG. 7. Brn-4 synergizes the effect of Cdx-2 in activating GLU-LUC expression. BHK fibroblasts were transfected with 2 µg of the indicated GLU-LUC fusion gene plasmid and the indicated amount (micrograms) of Brn-4 cDNA, Cdx-2 cDNA, or pCDNA3.1 (as vector). Cells were harvested for LUC reporter gene analysis 18 h after transfection. Relative LUC reporter gene activity was calculated as the fold increase in activity compared with that in cells that received the empty pCDNA3.1 plasmid transfection, which was defined as 1-fold (mean ± SE; n = 3).

 
To assess the contribution of the binding sites of Cdx-2 and Brn-4 within the G1 enhancer element, we generated the WT GLU (G1)-TK-LUC fusion gene construct and two mutants, carrying the mutation on either Cdx-2- or Brn-4-binding sites (Fig. 1AGo). These LUC fusion gene plasmids were transfected into BHK cells along with Cdx-2, Brn-4, or Cdx-2 plus Brn-4. Figure 8Go shows our representative reporter gene assay results. Mutating either Brn-4- or Cdx-2-binding sites completely blocked the activation by Brn-4 transfection and attenuated the activation by Cdx-2 transfection. Mutating either Cdx-2- or Brn-4-binding sites substantially attenuated, but did not abolish, the synergistic activation. Based on this result, we propose that the synergistic activation involves two mechanisms: cooperative DNA binding and mutual recruitment of Cdx-2 and Brn-4 (see details in Discussion). The second mechanism requires physical interaction between Cdx-2 and Brn-4.

Detection of physical interaction between Brn-4 and Cdx-2
The physical interaction between Cdx-2 and Brn-4 was examined using a GST-fusion protein pull-down assay, with GST-Brn-4 fusion proteins against Cdx-2 expressed in the COS-1 cell line. Our results are shown in Fig. 9Go. Although the WT GST-Brn-4 fusion protein was able to pull down Cdx-2 (lane 3), GST alone was not (lane 2). This indicates the existence of an interaction between Cdx-2 and Brn-4. Interestingly, the Brn-4 D1, D2, and D3 deletion fusion proteins were also found to pull down Cdx-2, indicating that the interaction domain in Brn-4 is located outside the transactivation and POU domains (lanes 4–6). The results shown in lane 7 suggest that the HD domain of Brn-4 is the region that interacts with Cdx-2, in agreement with recent observations that HDs in other HD proteins, such as Nkx2.5 and Tinman, play roles in their homodimerization and heterodimerization with other HD proteins (30, 31).


Figure 9
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  		FIG. 9. GST fusion protein pull-down assay shows the physical interaction between Brn-4 and Cdx-2. A, Approximately 200 µg total protein from Cdx-2-transfected COS-1 cell lysate was incubated with GST (lane 2) or a GST-Brn-4 fusion protein (lanes 3–7). After the pull-down assay, the presence of Cdx-2 protein was detected by Western blotting. Lane 1, Cdx-2 expression in Cdx-2-transfected COS-1 cell lysate. B, Coomassie blue staining shows the sizes of the GST and GST-Brn-4 fusion proteins used in the pull-down assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Shortly after the discovery of the homeobox genes in 1984, endocrinologists began to explore the involvement of these master control genes in regulating the expression of hormone-encoding genes in endocrine organs, including the pancreatic islets, and in the genesis of hormone-producing cells. It is well known that pancreatic islet-specific differentiation and gene expression require the functions of basic loop helix transcription factors such as neurogenin-3, ß2/NeuroD, Foxa proteins, as well as HD proteins from different families (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42). One important phenomenon is that HD protein transcription factors, like many other so-called tissue-specific transcription factors, are never expressed as specifically as their downstream target genes. In pancreatic islets, Pdx-1 has been found to regulate the expression of both insulin and somatostatin genes as well as several other B cell-specific genes (39). The paired HD protein Pax6, however, is expressed in three major types of hormone-producing endocrine cells in pancreatic islets, and it could be involved in regulating the expression of insulin, proglucagon, and somatostatin genes (33). More recent studies have shown that NKX6 HD proteins play roles in pancreatic development and both pancreatic A and B cell differentiation (10).

Previous studies have shown that both Cdx-2 (15) and Brn-4 (12) are able to bind to the G1 enhancer element of the proglucagon promoter and activate its expression. It is common for one HD protein to use other transcription factors, including other HD proteins, as cofactor. Considering the fact that the binding sites for these two HD proteins are closely located, we asked whether these two HD proteins activate proglucagon expression synergistically. For this purpose, we isolated hamster Brn-4 cDNA and examined the effect of Brn-4 and/or Cdx-2 on proglucagon expression. Either Brn-4 or Cdx-2 was shown to provoke endogenous proglucagon mRNA expression in the In111 cell line. In addition, ectopic expression of both of them further enhanced proglucagon mRNA expression in this cell line.

To examine the mechanisms underlying the redundant and synergistic activation, we localized the activation domain of Brn-4 at its N terminus. This is consistent with the observations for other POU proteins, including OCT-1 (43), Brn3b (44), and Pit1 (45), and the caudal HD protein Cdx-2 (46). We then demonstrated that Brn-4 itself is not a strong activator of the proglucagon promoter, that Brn-4 synergistically activates the expression of proglucagon promoter with Cdx-2, and that the activation domain of Brn-4 is not absolutely required for synergistic activation. These observations collectively suggest that Brn-4 serves as a cofactor for Cdx-2 in regulating proglucagon expression.

The existence of the Cdx-2 homeobox gene was initially identified by James and Kazenwadel in 1991 (47) in the examination of the expression of Hox genes in intestinal epithelia by RT-PCR using degenerate primers. The first complete Cdx-2 cDNA, however, was isolated by German et al. (27) in 1992 from the hamster insulin-producing cell line HIT. German et al. (27) demonstrated activation of the insulin I promoter and its enhancer element by Cdx-2 cDNA transfection in their early studies. We (48) recently reported activation of endogenous insulin mRNA expression and insulin hormone synthesis by Cdx-2 overexpression in the pancreatic RIN-1056A cell line. Cdx-2 was also found to be abundantly expressed in all proglucagon-producing pancreatic and gut endocrine cell lines, but not in the pancreatic In111 cell line, which does not express proglucagon mRNA (15). Cdx-2 binds to the proglucagon promoter G1 enhancer element and activates both proglucagon promoter and endogenous proglucagon mRNA expression in the InR1-G9 cell line (15, 18). In this study we demonstrated that ectopic expression of Cdx-2 in In111 cells provoked them to express endogenous proglucagon mRNA; this supported our belief that Cdx-2 is a critical transactivator for proglucagon expression. However, the role of Cdx-2 in regulating proglucagon expression and genesis of the pancreatic A cell lineage has not been confirmed using the in vivo knockout strategies. Cdx-2–/– mice are embryonic lethal, whereas Cdx-2+/– mice show multiple defects, with normal development of pancreatic islets (49). One possible explanation could be that this gene is haploinsufficient for selected tissues that do not include pancreatic islets (29). To verify the role of Cdx-2 in regulating proglucagon expression and pancreatic A cell development in vivo, a cell type-specific knockout approach is required.

Evidently, as mentioned above, one HD protein could be involved in regulating more than one target gene in pancreatic islets and elsewhere. In contrast, it is also true for the involvement of multiple HD proteins in regulating the expression of a given target gene. To date, scientists have demonstrated in their in vivo and/or in vitro experiments that more than a dozen HD proteins may play roles in regulating proglucagon expression and/or the genesis of pancreatic A cells, including members of the HOX/Hox, caudal, POU, LIM, Paired, and NKX families (10, 11, 12, 13, 15, 16, 17, 18, 20, 22, 23, 25, 26, 27, 28, 29, 48). Brn-4 belongs to the class III POU HD family and is highly expressed in neural stem cells. It regulates stem cell-specific genes, including the intermediate filament protein nestin. Mutations in this gene may lead to X-linked deafness (22). Its expression in pancreatic A cells, but not in B cells, and its binding to the proglucagon promoter G1 enhancer element and activation of its expression were first demonstrated by Hussain et al. (12) in 1997. Brn-4 expression starts on embryonic d 10 in the pancreas, just before the expression of Pax6. Both Pax6 and Brn-4 appear in the glucagon-immunoreactive cells. On embryonic d 19, no Brn-4 colocalization was observed with insulin or somatostatin, suggesting that Brn-4 is an A cell-specific transcription factor. In 2001, Wang et al. (25) showed that induced Brn-4 expression in the INS{alpha}ß cell line initiated detectable expression of glucagon without affecting B cell-specific gene expression. In 2002, Hussain et al. (13) showed that misexpression of Brn-4 by the Pdx-1 gene promoter resulted in ectopic expression of the proglucagon gene in insulin-expressing pancreatic B cells in mice. These observations strongly suggested the role of this master control gene in directing the development of A cell lineage. However, in homozygous Brn-4–/– mice, pancreatic bud formation, proglucagon-expressing cell numbers, and related physiological measurements all appeared normal (33).

The observations made in our in vitro studies provide potential explanations for normal proglucagon gene expression in Brn-4–/– and Cdx-2+/– mice. Cdx-2 and Brn-4 may have redundant functions on binding to the G1 element of the proglucagon gene promoter, recruiting the coactivators, and activating proglucagon gene expression. Furthermore, synergistic activation may still occur in Brn-4–/– mice due to the expression of a compensatory factor. For example, in the absence of Brn-4, the animal may overexpress other POU HD proteins, such as OCT-1, to serve as the cofactor of Cdx-2. We observed that OCT-1 can replace Brn-4 in synergizing with Cdx-2 in activating expression of the proglucagon promoter G1 element (data not shown). One may also speculate that the role of Cdx-2 could be replaced by a Hox or Hox-like HD protein that is expressed in pancreatic A cells.

To study at the molecular level how these two HD proteins produce synergistic effects on proglucagon promoter expression, we generated mutant constructs of GLU (G1)-TK-LUC. We found that mutating either Brn-4- or Cdx-2-binding sites substantially attenuated, but did not abolish, the synergistic activation. We propose that there are two mechanisms involving in such synergistic activation. The first is caused by cooperative DNA binding. In this case, the binding of either Brn-4 or Cdx-2 to the G1 enhancer element of proglucagon promoter facilitates the binding of DNA by the other party. This hypothesis requires both Cdx-2- and Brn-4-binding sites. Such a model has been suggested by TenHarmsel et al. (50), who studied the binding of even-skipped proteins to the ultrabithorax-proximal promoter in Drosophila. The second possibility is that Brn-4 may recruit Cdx-2 and vice versa. This hypothesis would explain the occurrence of moderate synergistic activation when one binding site is mutated. It will be interesting to use chromatin immunoprecipitation and other approaches to examine these models and to investigate whether these models have general applications for HD proteins.

In conclusion, via conducting a number of in vitro experiments, we have assessed redundant and synergistic effects of Cdx-2 and Brn-4 on regulating proglucagon promoter and the effect of ectopic expression of these two HD proteins on endogenous proglucagon mRNA expression in the In111 cell line. Our results collectively suggest that Brn-4 serves as a cofactor for Cdx-2 in regulating proglucagon expression and raise a challenging question about how to assess the synergistic and redundant roles of HD proteins in regulating gene expression.


   Acknowledgments
 
We thank Dr. Michael German for providing the original hamster Cdx-2 cDNA, and Winship Herr for providing the OCT-1 cDNA.


   Footnotes
 
This work was supported by grants from the Canadian Institute of Health Research (MOP36398; to T.J.) and the Canadian Diabetes Association (1198; to T.J.).

First Published Online December 22, 2005

Abbreviations: BHK, Baby hamster kidney; CMV, cytomegalovirus; GLP, glucagon-like peptide; HD, homeodomain; GST, glutathione-S-transferase; LUC, luciferase; PGDP, proglucagon-derived peptide; POU, Pit-1, Oct-1 and Oct-2, Unc-86; TK, thymidine kinase; WT, wild type.

Received July 20, 2005.

Accepted for publication December 13, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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